Podocyte depletion is a hallmark of progressive glomerulosclerosis and diabetic nephropathy. CD2- associated protein (CD2AP) is a widely-expressed cytoplasmic adaptor protein that has previously been implicated in podocyte slit diaphragm protein complex formation. CD2AP null mice develop progressive glomerulosclerosis early in life and CD2AP haploinsufficiency is associated with glomerular disease susceptibility in humans. However, it remains unclear how loss of CD2AP function may cause glomerulosclerosis. In this renewal application, we will extend our work to test the hypothesis that CD2AP promotes renal epithelial cell survival by functioning as a molecular switch that suppresses the TGF-beta receptor type I (Tgfbr1)/Smad3-dependent apoptosis pathway and activates a Tgfbrl/phosphoinositide-3- kinase (PI3K) -dependent survival pathway. If confirmed, our studies will provide a molecular mechanism to explain how loss of CD2AP may enhance podocyte apoptosis and depletion to promote glomerular disease in mice and men based on a novel, essential functional role for CD2AP as multi-pathway regulator in TbRI- induced signal transduction. The relevance of this work is further underscored by recent observations suggesting that in the absence of direct genetic defects, functional downregulation of CD2AP expression is a common finding in mouse models and human glomerular disease. Specific Aims: 1. Identify the molecular determinants and characterize the regulation of the TGF-p-inducible interaction of CD2AP and cytoplasmic Tgfbrl. We will a) determine the sequence motif and structural model of Tgfbrl that underlies the interaction with CD2AP, and b) compare it with the Smad-binding model mediated by the GS region and L45 loop of Tgfbrl. 2. Investigate how CD2AP mediates negative regulation of Tgfbr1/Smad3 signaling. We will examine whether a) CD2AP competes with SmadS for binding to activated Tgfbrl; and b) CD2AP targets internalized Tgfbrl complexes for degradation and termination of signaling. 3. Define the role of podocyte-specific TGF-b receptor signaling in podocyte apoptosis and depletion in vivo, and determine whether this podocyte-specific mechanism underlies the progression of glomerular injury to glomerulosclerosis in CD2AP-/- mice? We will determine in vivo whether a) conditional deletion of TGF-b receptor type II (Tgfbr2) in podocytes using cre/lox technology prevents podocyte apoptosis, depletion, and/or glomerulosclerosis in CD2AP-/- mice; and b) whether podocyte-specific, controlled activation of TGF-(3 receptor signaling is sufficient to induce podocyte apoptosis, podocyte depletion and glomerulosclerosis in vivo using doxycycline-inducible expression of constitutively active Tgfbrl (Tgfbrl (AAD)). [unreadable] [unreadable] [unreadable]